Our basic understanding of how DNA carries out its biological function has been based on the Watson-Crick double helix as the dominant functional form of duplex DNA. However, Watson-Crick base-pairs cannot explain many fundamental biochemical aspects of duplex DNA, including how proteins recognize DNA with high sequence-specificity; how Watson-Crick faces of nucleotide base-pairs can become appreciably solvent exposed and prone to chemical damage; how damaged base-pairs can be stably accommodated in DNA and recognized by repair enzymes; and how errors arise during replication, transcription, and translation. The main hypothesis in this proposal is that canonical Watson-Crick base-pairs and non-canonical mispairs can transiently adopt alternative, higher energy, and sparsely populated conformations that are difficult to detect and characterize by biophysical methods, and that these transient alternative base-pairs provide a new layer of structural and dynamic complexity that is employed to drive many important DNA functions. Aim 1 will develop methods for identifying and experimentally characterizing Hoogsteen base-pairs in X-ray structures of DNA that may have been improperly modeled as Watson-Crick base-pairs. This Aim will also test the hypothesis that Hoogsteen base-pairs play important roles in maintaining genome stability in structurally stressed environments and in sequence-specific DNA recognition by proteins. Aim 2 will test the hypothesis that Hoogsteen base-pairs provide a basis for exposing Watson-Crick faces of nucleotide bases for sequence-specific alkylation damage. It will also test the hypothesis that damaged bases can be stably accommodated in DNA as Hoogsteen base-pairs that induce DNA bending and play functional roles in recognition by repair enzymes. Aim 3 will develop methods to characterize transient Watson-Crick-like mispairs that are stabilized by rare tautomeric and anionic bases. We will test the hypothesis that anionic Watson-Crick-like G-T mispairs provide the basic mechanisms for spontaneous and damaged-induced G-T misincorporation during DNA replication. The proposed fundamental studies of DNA structure and dynamics will redefine our view of the iconic DNA double helix, and uncover a rich layer of mechanistic complexity hidden in unconventional base-pairs that have so far proven difficult to capture and characterize at atomic resolution.